Potassium channels (K channels) are critical components of electrical signaling, a basic biological process that is essential to the function of nerve and muscle. The channels themselves are gated pores; K ions flow through the pore when it is opened (by voltage or signaling molecules), producing an ionic current that electrically "relaxes" the nerve or muscle cell. The voltage- and calcium-activated "maxi-K" channel has served as a prototype for understanding K channel modulation and gating. In terms of physiology, maxi-K channels are especially important in the relaxation of vascular smooth muscle to regulate blood pressure. Despite our knowledge of the function of these channels, little is known of their structure or of the molecular basis of their function. It is hoped that a better understanding of maxi-K channel structure and function will ultimately lead to advances in the treatment of neurological and cardiovascular disease. The maxi-K channel, like other voltage-dependent K channels, undergoes a series of conformational changes when it gates. In this proposal, we aim to test and expand upon recent hypotheses of these gating movements, using a combination of patch-clamp recording and time-resolved fluorescence spectroscopy. Our patch-clamp and spectroscopy experiments will be performed on channels in intact, living cells, and can thus answer questions that cannot be addressed with crystallography. By detecting specific fluorescence quenching interactions between attached fluorophores and endogenous sidechains in the channel, we can obtain estimates of intermolecular distances, and thus gain insight toward channel structure. Our specific aims are: 1) to determine secondary structural features of the maxi-K channel by estimating distances between sidechains, using independent but complementary approaches of electrophysiology and fluorescence spectroscopy; 2) to determine the solvent accessibility of specific amino acid positions on the channel, by measuring excited-state lifetimes of fluorescently-labeled channels in the presence of iodide (an aqueous quenching agent); and 3) to locate amino acid positions that sense gating movements, by performing fluorescence spectroscopy on open and closed channels in patch-clamped whole cells. This combination of experiments will contribute to significant advances in our knowledge of K channel gating.